Systematic Comparison of High-Fat Diet- and Streptozotocin-Induced Prediabetic Obese C57BL/6J Mouse Models: A Sex-Based Protocol Optimization Study.

AIMS: Animal models of prediabetes (Pre-DM) are essential for studying metabolic disease and testing therapies, yet high-fat diet (HFD) and HFD-plus-streptozotocin (STZ) protocols vary in feeding duration, STZ dose, and diagnostic criteria and rarely account for sex. We compared five protocols in male and female C57BL/6J mice to find the best balance of duration, cost, and phenotypic stability and to characterize how HFD and HFD-STZ differ in their effects on glucose and lipid metabolism.

MATERIALS AND

METHODS

Five-week-old mice were stratified by sex and assigned to three HFD-only groups (HFD-12w, -16w, -20w) and two STZ groups of differing cumulative dose (HFD-STZ-L, -H). Body weight, food and energy intake, body fat percentage, fasting blood glucose (FBG), OGTT, ITT, fasting insulin, serum lipids, hepatic Oil Red O, histopathology, liver and kidney safety, and per-animal cost were assessed.

RESULTS

HFD induced obesity of equal magnitude in both sexes (non-significant Sex × Diet interactions for body weight and fat mass). Glucose and hepatic-lipid metabolism showed clear sex dimorphism: in males these deteriorated by Week 12, whereas females required 16 weeks to reach the Pre-DM threshold, at a higher preset STZ dose. HFD-STZ-L matched HFD-12w in both sexes within a shorter cycle (overall success 73.3%). Notably, STZ reversed the HFD lipid phenotype, synchronously reducing body fat, serum lipids, and hepatic fat in females.

CONCLUSIONS

HFD-STZ-L is preferred for glucose and β-cell studies in both sexes; HFD-12w preserves the intact female obesity phenotype; HFD-20w is preferred for MASLD studies. Biological sex must be incorporated into model design and phenotype interpretation.